The objective of this investigation was to develop quantitative isocratic HPLC methods for the analysis of digoxin and digitoxin. An HPLC system that employs a reverse-phase column, UV detection at 220 nm and solvent systems consisting of various proportions of water, methanol, isopropanol and dichlormethane was developed for the separation of digoxin, digitoxin and their potential degradation products and metabolites. HPLC separations of the above compounds by isocratic, solvent switchover and gradient elution modes were carried out in chromatographic times of 27, 16 and 13 minutes, respectively. For purposes of monitoring the separation of dihydro metabolites of digoxin, a 100% fluid recovery system was developed for use in the HPLC analysis of digoxin and its metabolites after fluorogenic post-column derivatization using the air-segmentation process. As an evidence of selectivity, the isocratic HPLC systems were utilized for the separation of a mixture of ten closely related steroids and the isolation of digitoxin from Digitalis purpurea leaf. The isocratic HPLC systems were found to be applicable for the quantitative analysis of digoxin and digitoxin in their respective dosage forms. The HPLC assay of digoxin and digitoxin dosage forms was carried out in less than forty-five minutes. These methods were found to be precise, accurate, sensitive enough for single tablet assay, and capable of simultaneously monitoring the potential degradation products or metabolites of digoxin and digitoxin. A comparison of the assay of digoxin and digitoxin dosage forms by HPLC and USP methods indicated that: (a) the precision and accuracy of both methods were comparable and within acceptable limits; (2) analysis by HPLC can be completed in less than forty-five minutes whereas the USP methods require over four hours; and (3) the HPLC methods have the advantages of higher sensitivity, selectivity and simplicity over the USP methods. The HPLC methods were used for the stability study of digoxin and digitoxin in their respective dosage forms. Lanoxin and digitoxin tablets were found to be stable under all the conditions of storage used in this study. Natigoxin tablets, Lanoxin injection and elixir were found to be subject to varying degrees and patterns of degradation. On the basis of the stability results it was observed that the assortment of pathways that may be involved at different conditions and times of storage'would make it difficult to estimate digoxin shelf-life from data obtained by accelerated aging. From the results of this investigation, it was concluded that the isocratic HPLC methods were suitable for the assay of digoxin and digitoxin dosage forms as well as for purposes of stability testing and simultaneous monitoring of degradation products or metabolites. This abstract represents the true contents of the thesis submitted. / Pharmaceutical Sciences, Faculty of / Graduate

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Estudos recentes sobre disfunção ventricular demonstram o potencial terapêutico da modulação da matriz extracelular. Isso se dá pela influência que a referida matriz tem sobre a função sistólica e a diastólica do coração. Outros estudos demonstram a influência do digital sobre os sistemas neurohormonais desbalanceados no cenário de disfunção ventricular levantando uma questão acerca do potencial do digital como modulador da deposição do colágeno intersticial e perivascular miocárdico. Sabendo-se da importância prognóstica que a concentração do colágeno no referido cenário tem, a literatura apresenta uma lacuna de conhecimento. Objetivo: Avaliar o papel do digital no remodelamento miocárdico em um modelo experimental. Material e Métodos: 60 ratos Wistar foram separados em 3 grupos de 20. Um grupo controle (GC); outro grupo submetido ao modelo experimental de uninefrectomia, administração de água de beber com 1% de NaCl e de aldosterona subcutânea (GA); e outro grupo submetido ao mesmo modelo experimental, mas também recebendo digitoxina na ração de comer na dose de 100 g/Kg/dia (GAD). Resultados: A fração de volume de colágeno intersticial e perivascular mostrou-se maior no GA comparado aos outros dois grupos (GC e GAD). O índice de desempenho miocárdico mostrou diferença estatisticamente significativa entre o GA (0,49 ± 0,08) e o GC (0,32 ± 0,06) e o GAD (0,4 ± 0,13) (p=0,001). Os níveis séricos de BNP mostraram diferença estatisticamente significativa entre o GA (1,07 ± 0,32 ng/ml) e o GC (0,75 ± 0,19 ng/ml) e o GAD (0,84 ± 0,21 ng/ml) (p=0,01). Os níveis de metaloproteinases não diferiram entre os grupos. Houve uma correlação positiva entre uma maior fração de encurtamento e menores níveis séricos de BNP no GAD. Conclusões: Esses dados demonstram que a digitoxina teve efeito reduzindo a deposição de colágeno intersticial e perivascular e melhorando a função cardíaca avaliada pelo BNP e IDM nesse modelo experimental / Recent studies on myocardial dysfunction are highlighting the therapeutic potential of the myocardial extracellular matrix management. Its interesting to highlight the importance of the dynamics of the cardiac extracellular matrix, because even the systolic and diastolic functions are implicated on it. Other studies showed that digital compounds may regulates the neuroendocrin misbalance due to myocardial impairment and influencing these systems the digital compounds may regulates the interstitial collagen deposition. Objective: To evaluate the role of the digital on a myocardial fibrosis in an experimental model, examining if the digital is able to prevent the collagen deposition. Methods: The sample was divided in 20 rats from the control group (CG); 20 rats submitted to a fibrosis experimental model in which the rats are uninefrectomized, drink water with 1% NaCl during the protocol and receive aldosterone through an osmotic minipump (AG); and 20 rats submitted to the same experimental model treated with digitoxin in a daily dose of 100 g/Kg (DAG). Results: The interstitial and perivascular collagen volume fraction showed a significant difference between the AG and the other 2 groups (CG and DAG). The myocardial performance index showed a significant difference between the AG (0.49 ± 0.08) and the CG (0.32 ± 0.06) and the DAG (0.40 ± 0.13) (p=0.001). The BNP levels showed a significant difference between the AG (1.07 ± 0.32 ng/ml) and the CG (0.75 ± 0.19 ng/ml) and the DAG (0.84 ± 0.21 ng/ml) (p=0.01). The metalloproteinases levels did not differ among the groups and there was a positive correlation between the shortening fraction and the BNP levels among the GAD animals. Conclusion: These data demonstrate the digitoxin positive effect on the myocardial collagen deposition in this experimental model of interstitial fibrosis and could have a new therapeutic target previously unexplored

Prostate cancer is the most occurring form of cancer in men in Sweden and new candidates for treatment towards advanced phases of prostate cancer needs to be investigated. One suggested treatment is vitamin D which will mediate effect via VDR and PDIA3 and cause cell cycle arrest.  Another treatment is digitoxin which will cause accumulation of intracellular Ca2+ leading to apoptosis. The aim of the project of was to investigate potential synergistic effects of 25-hydroxyvitamin D3 and digitoxin in prostate cancer cell lines and find out effects mediated by PDIA3. DU145 and LNCaP cells were seeded and treated with 25-hydroxyvitamin D3 (10-10, 10-9, 10-7 M), digitoxin (25 ng/ml and 50 ng/ml) and four combinations of 25-hydroxyvitamin D3 and digitoxin. Cell viability assay was performed for determining the number of viable cells. Treatment with 25-hydroxyvitamin D3 10-7M + digitoxin 50ng/ml (68%), 25-hydroxyvitamin D3 10-9M + digitoxin 25ng/ml (39%), 25-hydroxyvitamin D3 10-9M + digitoxin 50ng/ml (69%), digitoxin 25ng/ml (26%) and digitoxin 50 ng/ml (44%) was statistically significant with increased cell viability compared to untreated control in DU145 after 48h of treatment. Treatment with 25-hydroxyvitamin D3 10-7M (12%) and 25-hydroxyvitamin D3 10-9M (12%) was statistically significant with increased cell viability compared to untreated control in LNCaP after 24h of treatment. The conclusion based on results from this study is that a combination of digitoxin and 25-hydroxyvitamin D3 does not inhibit cell viability in DU145 or LNCaP cancer cell lines.

Estudos recentes sobre disfunção ventricular demonstram o potencial terapêutico da modulação da matriz extracelular. Isso se dá pela influência que a referida matriz tem sobre a função sistólica e a diastólica do coração. Outros estudos demonstram a influência do digital sobre os sistemas neurohormonais desbalanceados no cenário de disfunção ventricular levantando uma questão acerca do potencial do digital como modulador da deposição do colágeno intersticial e perivascular miocárdico. Sabendo-se da importância prognóstica que a concentração do colágeno no referido cenário tem, a literatura apresenta uma lacuna de conhecimento. Objetivo: Avaliar o papel do digital no remodelamento miocárdico em um modelo experimental. Material e Métodos: 60 ratos Wistar foram separados em 3 grupos de 20. Um grupo controle (GC); outro grupo submetido ao modelo experimental de uninefrectomia, administração de água de beber com 1% de NaCl e de aldosterona subcutânea (GA); e outro grupo submetido ao mesmo modelo experimental, mas também recebendo digitoxina na ração de comer na dose de 100 g/Kg/dia (GAD). Resultados: A fração de volume de colágeno intersticial e perivascular mostrou-se maior no GA comparado aos outros dois grupos (GC e GAD). O índice de desempenho miocárdico mostrou diferença estatisticamente significativa entre o GA (0,49 ± 0,08) e o GC (0,32 ± 0,06) e o GAD (0,4 ± 0,13) (p=0,001). Os níveis séricos de BNP mostraram diferença estatisticamente significativa entre o GA (1,07 ± 0,32 ng/ml) e o GC (0,75 ± 0,19 ng/ml) e o GAD (0,84 ± 0,21 ng/ml) (p=0,01). Os níveis de metaloproteinases não diferiram entre os grupos. Houve uma correlação positiva entre uma maior fração de encurtamento e menores níveis séricos de BNP no GAD. Conclusões: Esses dados demonstram que a digitoxina teve efeito reduzindo a deposição de colágeno intersticial e perivascular e melhorando a função cardíaca avaliada pelo BNP e IDM nesse modelo experimental / Recent studies on myocardial dysfunction are highlighting the therapeutic potential of the myocardial extracellular matrix management. Its interesting to highlight the importance of the dynamics of the cardiac extracellular matrix, because even the systolic and diastolic functions are implicated on it. Other studies showed that digital compounds may regulates the neuroendocrin misbalance due to myocardial impairment and influencing these systems the digital compounds may regulates the interstitial collagen deposition. Objective: To evaluate the role of the digital on a myocardial fibrosis in an experimental model, examining if the digital is able to prevent the collagen deposition. Methods: The sample was divided in 20 rats from the control group (CG); 20 rats submitted to a fibrosis experimental model in which the rats are uninefrectomized, drink water with 1% NaCl during the protocol and receive aldosterone through an osmotic minipump (AG); and 20 rats submitted to the same experimental model treated with digitoxin in a daily dose of 100 g/Kg (DAG). Results: The interstitial and perivascular collagen volume fraction showed a significant difference between the AG and the other 2 groups (CG and DAG). The myocardial performance index showed a significant difference between the AG (0.49 ± 0.08) and the CG (0.32 ± 0.06) and the DAG (0.40 ± 0.13) (p=0.001). The BNP levels showed a significant difference between the AG (1.07 ± 0.32 ng/ml) and the CG (0.75 ± 0.19 ng/ml) and the DAG (0.84 ± 0.21 ng/ml) (p=0.01). The metalloproteinases levels did not differ among the groups and there was a positive correlation between the shortening fraction and the BNP levels among the GAD animals. Conclusion: These data demonstrate the digitoxin positive effect on the myocardial collagen deposition in this experimental model of interstitial fibrosis and could have a new therapeutic target previously unexplored

<p>This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, <i>N</i>-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation.</p><p>Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from <i>Phoradendron tomentosum</i>. In addition, the cardiac glycoside digitoxin was isolated from <i>Digitalis purpurea</i>. All these substances expressed cytotoxicity and a neutrophil challenging activity.</p><p>Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC₅₀: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC₅₀ values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies.</p><p>The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC₅₀ 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.</p>

This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, N-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from Phoradendron tomentosum. In addition, the cardiac glycoside digitoxin was isolated from Digitalis purpurea. All these substances expressed cytotoxicity and a neutrophil challenging activity. Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC50: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC50 values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies. The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC50 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.